4.2 Four-arm oligoaminoamides design and synthesis
Four-arm oligoaminoamides were synthesized by SPAS using a common Fmoc strategy.
This method uses linkers attached to small porous beads, resins, for building peptide chains with consecutive coupling, washing, and deprotection steps. Amino acids are protected with protection groups that enable selective deprotection. The Fmoc base labile protection group is used as protection of a reactive amine that will react in the next coupling step, whereas other reactive groups such as thiol, hydroxy or additional amino groups are protected with other acid labile protecting groups. Standard steps in SPAS encompass resin loading, coupling, washing, deprotection, washing and subsequent cycle repeats. After every washing step, Kaiser’s test is performed to determine if the coupling, deprotection or washing steps were executed successfully.
Figure 16: Illustration of repeating coupling-washing-deprotection-washing steps in SPAS.
Firstly, the C-terminal amino acid is loaded on the resin and repeating cycle of coupling, washing, deprotection and washing steps is preformed to elongate the peptide chain. After coupling with all amino acids, obtained peptide is cleaved off the resin with TFA and the remaining protection groups are removed.
Its ability to remove excess reagents makes SPAS synthesis so convenient. It allows beads to be washed through a filter in the syringe reactor, preventing the loss of product due to immobilization of peptide on the resin beads that cannot permeate through the filter pores.
Using this technique, peptide structures can be synthesized very quickly, and with improving technology SPAS can also be automated. In addition to commercially available amino acids, other reagents are used in SPAS synthesis. PyBOP/HBTU and HOBt are both used as
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activation reagents, as well as suppressors of racemization. A non-nucleophilic base like DIPEA is used as a proton scavenger. DIPEA ensures both the deprotonation of the amine, as well as the carboxylic function, facilitating easier active ester formation with the activation reagent. As organic solvents, DCM and DMF are mainly used to create nonaqueous environment for successful coupling, while facilitating swelling of the resin during the reaction at the same time.(59) The final product was cleaved off the resin using a cleavage cocktail that includes TFA – a very strong acid that can also remove other remaining protection groups. During this process highly reactive cationic species are generated from the protecting groups and resin-linkers. These species can react and modify electron-rich amino acid functional groups, such as tyrosine, tryptophan, methionine, and cysteine. The appearance of cationic species called for adding various nucleophilic reagents, known as scavengers, to the cleavage cocktail: water is a moderately effective scavenger for t-butyl cations and the products of the cleavage of arylsulphonyl-based protecting groups;
EDT is the best scavenger for t-butyl cations and protects unprotected tryptophan against sulphonation, while also facilitating the removal of the trityl protecting group from cysteine:
TIS is very efficient at quenching highly stabilized cations liberated after cleaving Trt. (60) TFA was mainly used at 95 % to achieve successful deprotection of all protection groups and to cleave the oligoaminoamide off the resin. For example, the Trt protection group on histidine needs TFA of at least 50 %, and a concentration of up to 90 % must be used to remove protection groups like Boc, OtBu, tBu, Trt on cysteine.(61) Groups such as t-butyl-based protecting groups, Pmc and Pbf from arginine, and Trt groups from asparagine, glutamine, histidine, and cysteine often call for TFA at 95 % to obtain a fully side deprotected peptide.(60)
In the first part of synthesis, the branching cores were synthesized. Ala-Wang resin was used instead of 2-chlorotritylchloride resin due to thermal hydrolysis of the trityl ester bond at a higher temperature, that we used for coupling amino acids with an automated synthesizer.(62) Only 0.25 eq. of Fmoc-L-Lys(Fomc)-OH relative to the free
resin-bound amines were coupled at the first Figure 17: Graph of dibenzofluene-piperidine adduct absorption peak at 301 nm
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step to reduce the density of four-arm oligoaminomaides which are prone to aggregation at a high density.(63) The remaining free resin-bound amines were acetylated to prevent reacting with further reagents. Lysin was introduced two times as a symmetrical branching point, owing to two primary amines, to branch the structure into four-arm structures. Histidine was coupled after every branching point with the intent of fine-tuning the proton sponge activity.(40) After the branching cores were synthesized, the load was determined spectrophotometrically. By the Fmoc deprotection, Fmoc group reacts to dibenzofluene-piperidine adduct absorbance of which can be measured at the absorption peak of 301 nm and can give us a quantitative amount of the loaded resin beads.(56)
resin
Figure 18: Reaction scheme of dibenzofluene-piperidine adduct formation
Following sequences of four-arm structures were synthesized automatically; [Stp-H-R]3, [Stp-H]3R3, [Stp-H-W]3, [Sph-H]3Y3, [Sph-H]3W3, [Sph-H]3R3, [Sph-H-R]3, [Sph-H-K]3, [Sph-H-W]3, and [SphH]3 twice. For more efficient coupling, amino acids were coupled at 75 °C and additional coupling was executed. The Stp and Sph building blocks were introduced to improve buffering capacity, compaction of nucleic acid and subsequent transfection. The Sph should be superior because of one additional amino group.(44) Arginine and lysine were introduced to improve binding ability. Arginine showed to be superior in regard to DNA compaction in comparison to lysine.(64) Tryptophan and tyrosine were introduced to improve hydrophobic interaction and to stabilize polyplexes.(49–51) To these sequences, cysteines and lysines with azido moiety were coupled manually, due to the tendency of cysteines to crosslink to each other at higher temperatures, and the possibility
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of azido lysine causing an explosion in the automatic synthesizer’s microwave, because of the released gasses. The purpose of cysteine is to stabilize polyplexes through covalent disulfide cross-linkage that can be reduced in the reductive cytosol and facilitates the release of the payload into the cytosol.(40)
Table 8: Illustration of the synthesized four-arm structures and their ID numbers
Structure and topology
1494 1518
1495 1516
1514
1513 1519 1493
1515 606
1517
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Azido lysine was introduced at the ends of the four arms, for further attachment of shielding agents or ligands using the DBCO click-chemistry strategy. None of the synthesized oligoaminoamides have been synthesized before, except for 606 that has served as a “gold standard” due to its so far obtained best transfection results. The 606 oligoaminoamide was thus used for comparison with the novel four-arm sequences.(40) For the synthesis of four-arm oligoaminoamides, 1 % of Triton™ X-100 (v/v) was added to the DMF and DCM to reduce the surface tension, as well as to make reactive groups more accessible, and to wash the resin beads properly after every coupling and deprotection step.(63) Due to bad deprotection results of the Fmoc group in the past experiments by using only 20 % of piperidine, additional 2 % of DBU was added to the deprotection solution which is a much stronger base in comparison to piperidine, and 2 % is sufficient for successful deprotection.(65,66) All four arms were purified through SEC, which is a good purification method of choice due to its capability of changing the TFA salt into the HCl salt. The latter is more viable for in vivo and in vitro experiments, because the TFA salt might induce toxicity connected issues.(67) For this reason, both dialysis as well as HPLC are not an applicable method duo to the polycationic nature of OAA. The structures of purified oligoaminoamides were confirmed by 1H-NMR (See Appendix). Polyplexes were formed and evaluated regarding nucleic acid binding efficiency, size, and zeta potential.